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Updated: Jul 11, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Specificity of mtDNA-directed PCR-influence of NUclear MTDNA insertion (NUMT) contamination in routine samples and
Ana Goios1, Lourdes Prieto, António Amorim
1Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), R. Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. aalmeida@ipatimup.pt
Abstract:
Nuclear mitochondrial insertions (NUMTs) are sequences homologous to mtDNA, which are present throughout the human nuclear genome. The possibility that these sequences may be accidentally amplified in reactions directed to mtDNA has been raised and evaluated by different groups and by different means. Despite that, data is still missing on the specificity of PCRs in routine procedures in what concerns contamination with nuclear mtDNA insertions (NUMTs). In this work, we performed PCR sequencing reactions with primers directed either to mitochondrial or to NUMT DNA with different annealing temperatures and in different tissues. We observed that (a) contamination with NUMTs depends on the sample and tissue, and (b) employing routine techniques, there is no risk of co-amplification. Only when mtDNA is almost completely removed from the samples does the number of NUMT copies exceed mitochondrial sequences, i.e., only in samples with virtually no mtDNA, such as those resulting from preferential semen lysis, is there a risk of accidental amplification of NUMTs. We suggest that to evaluate a possible co-amplification of NUMT DNA, it is more relevant to take into account sample processing and original tissue of the samples, and consequently the relative proportions of NUMT and mtDNA, rather than the presence of NUMTs by itself, irrespectively of its proportion.
Insights
Nuclear mitochondrial insertions (NUMTs) are unlikely to contaminate routine mitochondrial DNA (mtDNA) PCRs. Accidental NUMT amplification risk is minimal unless mtDNA is virtually absent, as seen in specific sample types.
Area of Science:
- Genetics
- Molecular Biology
- Genomics
Background:
- Nuclear mitochondrial insertions (NUMTs) are nuclear DNA sequences homologous to mitochondrial DNA (mtDNA).
- Concerns exist regarding the potential for NUMTs to be co-amplified during PCR assays targeting mtDNA.
- The specificity of routine PCR procedures concerning NUMT contamination remains incompletely understood.
Purpose of the Study:
- To evaluate the specificity of PCR assays for mtDNA in the presence of NUMTs.
- To determine the risk of accidental NUMT co-amplification under routine laboratory conditions.
- To identify factors influencing NUMT amplification in PCR reactions.
Main Methods:
- PCR sequencing reactions were performed using primers specific to either mtDNA or NUMT DNA.
- Experiments utilized varying annealing temperatures and different human tissue samples.
- Relative proportions of NUMT and mtDNA were assessed in various sample types.
Main Results:
- Contamination with NUMTs is dependent on the specific sample and tissue origin.
- Routine PCR techniques demonstrated no significant risk of NUMT co-amplification.
- NUMT copies only exceed mitochondrial sequences when mtDNA is nearly absent, such as in samples from preferential semen lysis.
Conclusions:
- The risk of NUMT co-amplification in routine mtDNA PCR is generally low.
- Sample processing and the original tissue type are critical factors in assessing NUMT co-amplification risk.
- Evaluating the relative proportions of NUMT and mtDNA is more informative than simply detecting NUMTs.
